Human pluripotent stem cells (hPS) are expected to revolutionize the accessibility to a variety of human cell types. The possibility to propagate pluripotent human embryonic-derived stem (hES) cells and human induced pluripotent stem (hiPS) cells and subsequently differentiate them into the desired target cell types will provide a stable and virtually unlimited supply of cells for a range of applications in vivo and in vitro.
Liver failure and end-stage liver diseases are responsible for a huge amount of deaths around the world and is a major burden on the health care system. Liver transplantation remains the most successful treatment. However, the efficacy of this procedure is limited and connected to many complications such as infection or rejection. Liver transplantation also suffers from shortage of available donor organs and the treated patients will very often be referred to lifelong immunosuppression therapy. By reducing the need for organs, cell-based treatment will be of great importance to both society and to the individuals suffering from these severe diseases.
Furthermore, the liver is the centre of metabolism and detoxification in the human body, and therefore huge efforts have been undertaken in order to identify a reliable source of functional cell types for in vitro testing. Unfortunately, the complexity and function of the liver is not mirrored by any cell type available today. The availability of primary human liver cells is very limited and the cells are also known to rapidly loose their normal phenotype and functional properties when used for in vitro applications. One often used alternative to primary cells are hepatic cell lines which in turn contain very low levels of (or totally lack) metabolising enzymes and have distributions of other important proteins substantially different from the native hepatocyte in vivo. Thus, many tests are still performed using animal material, even though liver metabolism is known to be species specific and thereby generating difficulties in predicting liver metabolism and toxicity in other species than the one tested.
In pharmaceutical development, adverse liver reactions remain the most prominent side effect. Therefore early prediction of human liver toxicity liabilities is of paramount importance when selecting compounds to enter clinical trials. Efforts to improve capabilities in this area must address both the availability question and development of models, which provide greater coverage for the complex biological processes which coincide to induce adverse liver injury in humans.
Accordingly there is an urgent need for a model system that mimics human liver cells and that is able to predict effects of candidate molecules in the development of new drugs or chemicals. Regarding both availability and physiological relevance, hPS cells may serve as an ideal renewable source of functional human hepatocytes.
During the embryogenesis and the formation of the yolk sac and the placenta, two types of endoderm cells form: the extraembryonic endoderm cells and the definitive endoderm (DE) cells. Extraembryonic endoderm arises at the blastocyst stage and eventually forms two subpopulations: visceral endoderm and parietal endoderm. Extraembryonic endoderm cells share the expression of many genes with definitive endoderm (DE) cells (cells that give rise to the endodermal organs), including the often analyzed transcription factors Sox17 (Kanai-Azuma et al., 2002), FoxA1 and HNF3b/FoxA2 (Belo et al., 1997; Sasaki and Hogan, 1993). However, some markers are expressed in both mesoderm and definitive endoderm, such as CXCR4. Those commonly expressed, markers can be used in combination with Sox17 and FoxA2 to type DE cells. Sox7 is a marker only expressed in extraembryonic endoderm.
The definitive endoderm cells give rise to endodermal organs and thus hepatic cell types. However, early endoderm development is not well understood. Directed studies of cultured mouse embryos (Lawson et al., 1986, 1991; Lawson and Pedersen, 1987) have revealed that DE begins to form at the embryonic days 6-6.5 (E6-6.5) and that by the end of gastrulation (E7.5), some cells only give rise to endodermal derivatives. It is not known whether the initial DE cells are multipotent. Fate mapping studies (Lawson et al., 1991; Tremblay and Zaret, 2005) suggest that the first endoderm cells that migrate through the primitive streak (PS) at E6.5 are fated to become liver, ventral pancreas, lungs and stomach. Co-culture experiments show that the endoderm at this state is not fully committed at the early state of development (Wells and Melton, 2000).
For in vitro purposes, for example D'Amour et al. and Hay et al. have developed protocols for deriving definitive endoderm from hES cells (D'Amour et al., 2005; D'Amour et al., 2006, Hay et al., 2007; Hay et al., 2008) as well as protocols for derivation of hepatic endoderm from hiPS cells (Hay et al. 2010).
The Wnt pathway describes a series of events that occur when Wnt proteins bind to cell-surface receptors of the Frizzled family, causing the receptors to activate other proteins and ultimately resulting in a change in the amount of β-catenin that reaches the nucleus. A membrane-associated Wnt receptor complex will, when activated by Wnt binding, inhibit a second complex of proteins that includes e.g. the proteins GSK3 and axin. This complex normally promotes the proteolytic degradation of the β-catenin intracellular signalling molecule. After this inhibition, a pool of cytoplasmic β-catenin is present intracellularly, and some β-catenin is able to enter the nucleus and interact with transcription factors to promote specific gene expression. The Wnt/β-catenin signalling regulates key physiological events inherent to the liver including development, regeneration and development of cancer, by dictating several biological processes such as proliferation, apoptosis, differentiation, adhesion, zonation and metabolism in various cells of the liver (Nejak-Bowen et al., 2008).
Crosstalk between mesoderm and endoderm is required for liver differentiation. The wnt signalling molecule, Promethues/Wnt2b has been shown to be expressed in the discrete lateral plate mesoderm adjacent to the endoderm that will become hepatic endoderm in the zebra fish. Morpholino antisence knock-down of the wnt-gene will obliterate or reduce the early hepatic differentiation markers of the hepatic endoderm, hHEX and Prox1, indicating a role for wnt signalling in hepatic cell fate specification. In addition inhibition of beta-catenin signalling in the zebrafish embryo strongly reduced the development of hepatic tissue, suggesting a role for the canonocal/beta-catening Wnt signalling in liver cell fate specification. (Ref. Elke Ober et al. Nature 442, 688-691(10 Aug. 2006) However, observations in xenopus suggests that wnt signalling is crucial for patterning of the definitive endoderm into the anterior and posterior endoderm, where inhibition of wnt signalling leads to the anterior endoderm and hepatic induction while wnt signalling leads to posterior endoderm and intestinal induction. However, just after anterior patterning wnt signalling is important for hepatic induction and delamination of hepatoblasts from the hepatic endoderm. The data from the zebra fish and xenopus are somewhat contradictory and represent the complexity of Wnt-signalling in early hepatic differentiation. Fine tuning and timing of non active and active Wnt signalling seem to be important for early hepatic differentiation.
The use of GSK inhibitors have previously been described for early differentiation towards endoderm. WO08094597 (Dalton) describes a method of producing mesendodermal from primate pluripotent stem cells (pPSC) by contacting the pPSC with an effective amount of GSK inhibitor in a differentiation media.
WO2007050043 (Stanton) describes a method for producing a mesodermal or an endodermal cell from a pluripotent stem cell, comprising a Wnt-signalling pathway in the pluripotent stem cell.
US2006003446 (Keller) describes a way of making a cell population enriched for endoderm cells culturing embryonic stem cells in the absence of serum and in the presence of activin and an inhibitor of Wnt-signalling.
US 20100062527 (Pera et al.) describes in Example 3 culturing of HES2 or 3 cells “for 5 days on MEF feeders with 20% FCS hES medium in organ culture dishes. 20% FCS hES medium was replaced with 3i medium and cells were keeping in culture in this medium for 3 days. Cells were detached as clumps by collagenase and feeder cells were removed by sedimentation in DMEM/F-12 medium. Cells were seeded on Matrigel-coated organ culture dish as 1 to 1 split and culture with 3i medium. After 3 to 5 days in culture hepatoblast-like cells appeared. They were subsequently propagated using 3i of Kubota's medium following enzymatic dissection.” The 3i medium contains: Neural basal medium 50%, DMEM/F-12 50%, N2 supplement 1/200 v/v, B27 supplement 1/100 v/v, 100 mM L-glutamine 1/100 v/v, 0.1 M beta-ME 1/1000 v/v, SU5402 (FGFR inhibitor) 2 μM, PD184352 (ERK cascade inhibitor) 0.8 μM, and CHIR99021 (GSK3 inhibitor) 3 μM.